Biological and chemical assays and processes that are used analytically or preparatively for research, clinical, diagnostic and industrial purposes often require fixation, or immobilization, of a functional substance onto a solid support (or substrate). This fixation often improves the stability and versatility of the substance without compromising its effectiveness and activity, and enables repeated usage of the substance. For example, functional substances which include biological substances such as enzymes, are typically immobilized on an inert support like silica or polyacrylamide gel to improve their stability against changing pH or temperature conditions when used in enzyme-catalyzed industrial processes, and to facilitate their subsequent separation from the reaction products. This enables re-use of the immobilized enzymes and significantly facilitates product purification, which leads to more cost-effective processes.
Immobilization of a functional substance including a biological substance may be effected by physical immobilization or chemical immobilization. One form of physical immobilization is physical adsorption (physisorption), where the functional or biological substance is attached to the substrate via encapsulation or electrostatic, hydrophobic or Van der Waals forces. Whilst physical adsorption provides a relatively simple immobilization method with wide applicability to a whole range of functional and/or biological substances, it often does not provide a sufficiently stable immobilization and is susceptible to leaching of the immobilized functional and/or biological substances.
A more stable method of immobilization of functional and/or biological substances is chemical immobilization, which covalently binds the functional and/or biological substance to the substrate as a result of a chemical reaction. Chemical immobilization typically results in improved activity, reduced non-specific adsorption, and higher stability of the functional and/or biological substance. However, chemical immobilization generally requires the chemical modification of the functional and/or biological substance or the substrate to promote their efficient binding.
Modification of the surface of a solid support material, or “pre-activation” of a solid support, to improve its binding to a functional and/or biological substance, typically involves the incorporation of reactive chemical moieties onto the surface of the generally poorly reactive polymeric material. Surface modification can be achieved by physical means, such as non-covalent attachment of an affinity spacer, or chemical means such as glutaraldehyde activation, cyanogen bromide activation, bromoacetylation, diazotation, ionizing-radiation induced oxidation and chemical grafting.
The non-covalent attachment of an affinity spacer is, however, associated with poor reproducibility and/or unstable binding to the surface of the substrates. Some covalent attachments, most noteworthy imines, but to a lesser extent also esters, can be hydrolyzed under the reaction conditions used for enzymatic reactions, resulting in partial loss of immobilized enzyme and leakage of enzyme into the reaction medium. Such problems may affect, amongst others, immobilization methods based on glutaraldehyde activation and bromoacetylation. Whilst diazotation, cyanogen bromide activation, ionizing-radiation induced oxidation, and chemical grafting produce covalent bonds which are more stable than non-covalent bonds, these methods involve the use of hazardous, expensive, complicated, and/or harsh reaction conditions.
Some of these methods also result in a high net charge on the solid support, which causes undesirable non-specific electrostatic binding of the functional and/or biological substance during subsequent procedures in a biological/chemical process. Another common problem encountered with the use of harsh reaction conditions is the unfavorable modification of surface properties, which may hamper the attachment of a functional and/or biological substance, particularly a large polymeric substance. This can lead to low loading of the functional and/or biological substance onto the substrate. Yet other problems encountered with some commercially available activated solid supports are low stability, pronounced toxicity and a lack of biocompatibility, resulting in short shelf life, difficult handling, and limited applicability for medical purposes.
Some of these methods rely on the further modification of “pre-activated” supports with an epoxysilane coupling agent for the immobilization of hydrophilic molecules. Other methods rely on preparation of a substrate by reacting a bisepoxyoxirane linker to immobilize a molecule to the substrate. The aliphatic linkers used in these methods lead to a decrease in the amount of reactive groups available for immobilization, a decrease in biocompatibility and a decrease in reproducibility.
There is a need to provide methods of preparing a substrate for immobilization of functional and biological substances that overcome, or at least ameliorate, one or more of the disadvantages described above.
There is a need to provide methods that are convenient, inexpensive, robust, and reliable for preparing a substrate for immobilization of functional and biological substances.
There is also a need to provide substrates which are stable, easy to handle, inexpensive, non-toxic, biocompatible and bio-degradable for immobilization of functionally and biologically active substances, and which can be used for immobilization of a wide range of substances at high loading densities with improved activity and reactivity.